Test Realisation

Cytotoxicity, MEM extracts, MTT test system, 24 hrs.

Cytotoxicity, MEM extracts, MTT test system, 24 hrs.
The procedures in this document should be considered as a summary of the corresponding standard operating procedure of the test house, indicating and validating any deviation from this standard operating procedure.

General Guidelines
EN-ISO 30993-5 (page 2-6)

Statement of purpose
The test system is applied to obtain qualitative and quantitative data regarding cytotoxic effects of the test material towards the cell metabolism of fibroblasts.

Sterilisation procedure
The test material is supplied sterilised. All procedures described are performed under sterile conditions.

Extract Preparation
The shaft of the needle is cut from the needle and not included in the extraction procedure.
Amounts of 4 grams test material and control materials are extracted (or a similar surface / volume ratio) at 37ºC for 24 hours in 20 ml cell culture medium (with 10 % fetal calf serum)
Negative control : UHMW Poly Ethylene
Positive control   : Para rubber

Procedure
A monolayer of cells (mouse lung fibroblasts, L929) is grown to 70 - 80% confluency, which is examined and scored microscopically. Then cells are challenged with an extract of the test material (n=6), the negative control material (n=6), the positive control material (n=6), or with control medium only (n=6). After exposure to the extract for 24 hours at 37ºC, the medium
  is removed, leaving a film of medium in each well, and the cells are examined and scored microscopically for cytotoxic effects: confluency of monolayer (table 1) and change of cellular morphology (table 2). The scores are corrected for the negative control, resulting in the microscopic average. MTT, a water soluble tetrazolium salt yielding a yellowish solution, is added to each culture being assayed. MTT is converted into an insoluble purple formazan dye by mitochondrial dehydrogenase enzymes.

Only active mitochondrial dehydrogenases of living cells will convert MTT into the insoluble purple formazan dye. The cultures are incubated for 3 hours at 37ºC. After introduction of absolute isopropanol, cells are lysed and the precipitated formazan is dissolved. Formazan concentrations are determined by measuring the optical density (OD) at 570 nm with background correction of the OD at 690 nm. The mean OD570 value obtained for the negative control is standardised as 0% inhibition. The mean OD 570 value of the positive control is standardised as 100% inhibition. The mean OD570 value of the mean score for microscopic change and the mean score for growth inhibition.

Acceptance Criteria
Cytotoxic response Cytotoxic effect Pass / fail
0.0 - 1.0 non toxic pass
1.1 - 3.0 slight toxic pass
3.1 - 5.0 mild toxic retest
5.1 - 7.0 moderate toxic fail
7.1 - 8.0 severe toxic fail

In case a retest is indicated, an End-Point Dilution cytotoxicity test including determination of cell growth inhibition is advised.

Criteria for test-validation.
The maximum conversion of MTT is obtained for incubation with control medium.
The test shall be considered valid when the following criteria are met: The negative control demonstrates an inhibition < 10% of the maximum conversion.
The positive control demonstrates an inhibition > 80% of the maximum conversion.

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